1. Field of the Invention
This invention relates to a process for purifying polypeptides of interest from microbial fermentation broth or homogenate. More particularly, a precipitation agent is introduced to the broth or homogenate to effect, for example, protein, DNA, and cell debris removal.
2. Description of Related Art
The advent of recombinant technology now allows for the production of high levels of proteins within suitably transformed host cells. As a result, there is increased demand for fast, robust, and efficient purification methods to recover the recombinantly produced proteins. Generally, proteins are produced by culturing cells, such as mammalian, insect, fungal, and bacterial cell lines, engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For polypeptides produced in mammalian cells, the purification scheme is significantly easier than for polypeptides produced in other types of host cells. Mammalian cells export the polypeptides so that they can be collected from the growth media, where they are present in relatively pure form. However, if the polypeptide is produced in a non-mammalian cell, e.g., a microorganism such as fungi or E. coli, the polypeptide will be recovered inside the cell or in the periplasmic space (Kipriyanov and Little, Molecular Biotechnology, 12: 173-201 (1999); Skerra and Pluckthun, Science, 240: 1038-1040 (1988)). Hence, it is necessary to release the protein from the cells to the extracellular medium by extraction such as cell lysis. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration.
Cell lysis is typically accomplished using mechanical disruption techniques such as homogenization or head milling. While the protein of interest is generally effectively liberated, such techniques have several disadvantages (Engler, Protein Purification Process Engineering, Harrison eds., 37-55 (1994)). Temperature increases, which often occur during processing, may result in inactivation of the protein. Moreover, the resulting suspension contains a broad spectrum of contaminating proteins, nucleic acids, and polysaccharides. Nucleic acids and polysaccharides increase solution viscosity, potentially complicating subsequent processing by centrifugation, cross-flow filtration, or chromatography. Complex associations of these contaminants with the protein of interest can complicate the purification process and result in unacceptably low yields.
As such, more selective means of releasing intracellular proteins facilitates further downstream processing. Several techniques have been reported to permeabilize cells and/or to extract intracellular proteins. These methods include the use of solvents, detergents, chaotropic agents, antibiotics, enzymes, and chelating agents to enhance cell permeability and/or promote extraction. Additions of certain compounds, such as glycine, to the fermentation medium during culture growth have also been reported to promote release of certain intracellular enzymes. Finally, techniques such as freeze-thaw treatment or osmotic shock have also been shown to release subsets of intracellular proteins.
However, these techniques are not necessarily applicable to all intracellular microbial proteins, and all have limited application for large-scale processing, and/or other disadvantages. For example, while solvents such as toluene and chloroform promote release of intracellular proteins, these substances are known to be toxic and/or carcinogenic (Windholtz et al., The Merck Index 10th Edition: 300 and 1364 (1983)). Ionic detergents, such as SDS, often irreversibly denature isolated proteins. Although non-ionic detergents are not normally denaturing, the recovered proteins are often associated with detergent micelles that can require additional processing to yield detergent-free protein. Chaotropic agents, such as urea and guanidine hydrochloride, can be denaturing at the concentrations required for complete release, and their effectiveness may be dependent on the growth phase of the culture. The use of lysozyme, which provides for a relatively gentle means of protein release, is limited because of its relatively high cost and because of the subsequent need to purify the protein of interest from the enzyme reagent. In addition, chelating agents, often used to enhance the effectiveness of other permeabilizing/release techniques such as lysozyme or toluene extraction, suffer from the disadvantage of non-specific release of host proteins.
Other methods for protein release also have disadvantages. For example, osmotic shock, in which cells are suspended in a high osmolarity medium, recovered, and subsequently placed in a low osmolarity buffer, requires additional processing steps with respect to other extraction alternatives (Moir et al., Bioprocess Technology, Asenjo eds: 67-94 (1990)) or necessitates the handling of large liquid volumes at low temperatures. This renders the method unattractive for large-scale processing.
Freeze-thaw treatment also releases intracellular proteins, although relatively low yields often result in multiple cycles or additional processing requirements. In addition, cell paste freezing is an added non-trivial processing requirement compared with other extraction alternatives.
Finally, reagents, such as glycine, have been added during fermentation to promote protein release to the extracellular medium (Aristidou et al., Biotechnology Letters 15: 331-336 (1993)). While partial release of several intracellular proteins has been reported, this approach requires direct coupling of fermentation and release strategies and subsequent separation of the protein of interest from a potentially complex extracellular broth.
Once the polypeptide of interest is released from the host cell, purification thereof from other cell components is required. Unfortunately, most extraction approaches, such as cell lysis, not only expose the protein to potential degradation by host cell proteases, but also make isolation of the protein from other elements of the resulting suspension more difficult. For example, the presence of negatively charged molecules, such as DNA, RNA, phospholipids, and lipopolysaccharides (LPS), often requires the use of anion-exchange chromatography (Sassenfeld, TIBTECH, 8: 88-93 (1990); Spears, Biotechnology, vol. 3—Bioprocessing, Rehm eds:40-51 (1993)) and/or precipitation with polycations, such as protamine sulfate (Kelley et al., Bioseparation, 1: 333-349 (1991); Scopes, Protein Purification Principles and Practice, 2nd edition, Cantor eds., pp. 21-71 (1987)), streptomycin sulfate (Wang et al., eds, Fermentation and Enzyme Technology: 253-256 (1979)), polyethylenimine (PEI) (Kelley et al., supra; Sassenfeld, supra; Cumming et al., Bioseparation, 6: 17-23 (1996); Jendrisak, The use of polyethyleneimine in protein purification. Protein purification: micro to macro, ed. Alan R., Liss, Inc, 75-97 (1987); Salt et al., Enzyme and Microbial Technology, 17: 107-113 (1995)), and/or aqueous two-phase extraction with immiscible polymer systems such as polyethylene glycol (PEG)/phosphate or PEG/dextran (Kelley et al., supra, Strandberg et al., Process Biochemistry 26: 225-234 (1991)).
Alternatively, the protein of interest may be precipitated away from non-proteinaceous polyanionic contaminants through the addition of a neutral salt such as ammonium sulfate or potassium chloride (Wheelwright, Protein Purification: Design and Scale up of Downstream Processing: 87-98 (1991); Englard et al., Methods in Enzymology Volume 182, Deutscher eds.: 285-300 (1990)) and/or a polymer such as PEG or dextran sulfate (Wang et al., supra; Wheelwright, supra). Where the protein of interest is positively charged, it will tend to bind to any negatively charged molecules present thereby, making purification of the protein virtually impossible.
Typically, researchers have utilized the initial fractionation steps, described above, to separate the offending polyanions from the protein of interest. Unfortunately, each of these initial separation methods suffers from severe disadvantages, especially when used in the manufacture of pharmaceutical reagents. For example, the large quantities of non-proteinaceous polyanionic contaminants found in bacterial lysates tend to reduce the binding capacities of anion-exchange chromatography resins. In addition, regeneration protocols are often rendered ineffective due to tenacious binding of the polyanions to the resins (Spears, supra). Finally, the low ionic strength conditions that favor protein binding are ineffective at disrupting polyanion-protein interactions and result in a lack of separation (Scopes, Protein Purification Principles and Practice, 3rd edition, Cantor eds., p. 171 (1994)). Protamine sulfate preparations are plagued by concerns over protease and viral contaminations. Moreover, unwanted protein precipitation can occur using this reagent (Scopes, Protein Purification Principles and Practice, 2nd edition, Cantor eds., 21-71 (1987)).
In the processing of pharmaceutical proteins, streptomycin sulfate is generally not used due to general apprehension over the use of antibiotics as process reagents (Scawen et al., Handbook of Enzyme Biotechnology 2nd edition, Wiseman eds.: 15-53 (1985)). PEI preparations are often contaminated with varying amounts of the ethylenimine monomer, a suspected cancer agent (Scawen et al, supra). PEI also tends to bind irreversibly to many chromatography resins, thereby limiting their effectiveness and the number of potential chromatography resins available for use post-PEI clarification. In general, aqueous two-phase extractions systems are difficult to predict and often require an empirical approach for determining conditions that move the protein of interest into the appropriate aqueous phase (Kelley et al., supra).
Techniques that specifically precipitate the protein of interest often result in the entrapment of the non-proteinaceous contaminants in the precipitate, rendering the separation ineffective (Scopes, supra; Wheelwright, supra).
Examples of patents describing protein recovery and purification include the following:
U.S. Pat. No. 5,665,866 discloses a process for obtaining antibodies in soluble and correctly folded and assembled form. It comprises a step to raise the operating temperature to from 34 to 60° C. at a time in the process selected to facilitate the subsequent isolation of soluble, correctly folded and assembled antibody, substantially free of other antibody-related material.
U.S. Pat. No. 5,760,189 discloses a method for releasing a thioredoxin-like fusion protein from E. coli, including negatively charged non-proteinaceous material, into a solution by adding chelator to the solution, and precipitating the negatively charged non-proteinaceous material from the solution by adding a divalent cation/alcohol solution to the solution to form a first soluble fraction containing the protein and a first insoluble fraction containing unwanted contaminants. Optionally, the temperature prior to the addition of chelator may be substantially cooler than after the addition of chelator. The divalent cation includes, for example, magnesium, manganese, and calcium, alone or in combination.
U.S. Pat. No. 5,714,583 discloses methods for the purification of factor IX in a solution comprising the steps of applying the solution containing factor IX to an anion-exchange resin, washing the anion-exchange resin with a solution having a conductivity that is less than required to elute factor IX from the resin, eluting the anion-exchange resin with a first eluant to form a first eluate, applying the eluate to a heparin or heparin-like (e.g., negatively charged matrix) resin, eluting the heparin or heparin-like resin with a second eluant to form a second eluate, applying the second eluate to an hydroxyapatite resin, and then eluting the hydroxyapatite resin with a third eluant to form a third eluate containing the purified factor IX.
U.S. Pat. No. 6,322,997 discloses a method for recovering a polypeptide comprising exposing a composition comprising a polypeptide to a reagent that binds to, or modifies, the polypeptide, wherein the reagent is immobilized on a solid phase; and then passing the composition through a filter bearing a charge that is opposite to the charge of the reagent in the composition, so as to remove leached reagent from the composition.
U.S. Pat. No. 6,214,984 discloses low-pH hydrophobic interaction chromatography (LPHIC) for antibody purification. In particular, the patent provides a process for purifying an antibody from a contaminant that comprises loading a mixture containing the antibody and the contaminant on a hydrophobic interaction chromatography column and eluting the antibody from the column with a buffer having a pH of about 2.5-4.5. Usually, the mixture loaded onto the column is at about the same pH as the elution buffer.
U.S. Pat. No. 6,121,428 provides a method for recovering a polypeptide comprising exposing a composition comprising a polypeptide to a reagent that binds to, or modifies, the polypeptide, wherein the reagent is immobilized on a solid phase; and then passing the composition through a filter bearing a charge that is opposite to the charge of the reagent in the composition, so as to remove leached reagent from the composition.
U.S. Pat. No. 5,641,870 provides a process for purifying an antibody, wherein a mixture containing the antibody and contaminant is subjected to LPHIC optionally at low salt concentration. The antibody is eluted from the column in the fraction that does not bind thereto. In the extraction step, frozen cell pellets are re-suspended at room temperature in 20 mM MES buffer, pH 6.0 containing 5 mM EDTA and 20 mM 4,4′-DTP previously dissolved in ethanol (3 liters of buffer/kg of cell pellet). The suspended cells are disrupted by two passages through a Mantin Gaulin homogenizer at 5500 to 6500 PSI. The homogenate is adjusted to 0.25% (v/v) with polyethyleneimine (PEI) and diluted with an equal volume of 2-8° C. purified water. The diluted homogenate is then centrifuged. The antibody fragment is found in the supernatant.
Historically, immunoglobulin G (IgG) has been purified from human serum and plasma (Putnam, ed., The Plasma Proteins, vol. 1 (Academic Press, 1975)). The purification process has often contained one or more precipitation steps. The most commonly used precipitation scheme for recovering IgG is the Cohn fractionation (Cohn et al., J. Amer. Chem. Soc., 72: 465 (1950)). However, other precipitation techniques have been reported (Niederauer and Glatz, Advances in Biochemical Engineering Biotechnology, v. 47 (Springer-Verlag Berlin Heidelberg, 1992); Sternberg and Hershberger, Biochim. et Biophys. Acta, 342: 195-206 (1974)). The pioneering work of purifying IgG from plasma using 6,9-diamino-2-ethoxyacridine lactate (USAN name and herein called ethacridine lactate and also known by the names ETHODIN™ or RIVANOL™), a highly aromatic cationic dye, is reported by Horejsi and Smetana, Acta Med. Scand., 155: 65 (1956). The following decade produced a number of publications showing the capability of 6,9-diamino-2-ethoxyacridine lactate to purify IgG and other proteins (Miller, Nature, 184: 450 (1959); Steinbuch and Niewiarowski, Nature, 186: 87 (1960); Neurath and Brunner, Experientia, 25: 668 (1969)) from biological materials, e.g., plasma and growth media. Use of ethacridine lactate to recover antibodies and other proteins from other sources has been reported. See Tchernov et al., J. Biotechnol., 69: 69-73 (1999); SU 944580 published 28 Jul. 1982; Franek and Dolnikova, Biotech-Forum-Eur, 7: 468-470 (1990); EP 250288 published 23 Dec. 1987; DE3604947 published 20 Aug. 1987; Rothwell et al., Anal. Biochem., 149: 197-201 (1985); Lutsik and Antonyuk, Biokhimiya, 47: 1710-1715 (1982); and Aizenman et al., Mikrobiol-Zh., 44: 69-72 (1982).
The primary step of recovering polypeptides from microorganisms is most often concerned with removing solid material, e.g., cells and cellular debris. It is important to recognize the need to separate the desired product from components present in conditioned medium with which it specifically interacts. Where the protein of interest is positively charged, it will tend to bind to any negatively charged molecules present, thereby making purification of the protein by traditional methods very difficult. Additional removal of contaminating soluble protein from crude microbial extracts, e.g., E. coli homogenate, during this step would simplify subsequent chromatography steps. Such additional removal would be especially valuable for industrial-scale production, resulting in decreased chromatography column size and production times.